The synthesis of inhibitor GPI2350 specific for the mammalian plasma membrane GPI-PLC was based upon the background knowledge about the (crystal) structure, substrate requirements, GPI recognition and cleavage mechanisms of bacterial and trypanosomal (G)PI-PLC as well as on inhibitors described for them so far. For trypanosomal GPI-PLC, GPI and PI are efficient and poor substrates, respectively, whereas the opposite is true for the bacterial PI-PLC. The latter has a region of protein sequence similarity to trypanosomal GPI-PLC of 80 residues (“Kuppe et al. (1989) J. Bacteriol. 171: 6077-6083”). For analysis of GPI recognition, the three-dimensional structure of PI-PLC from B. cereus has recently been determined at 2.2 A resolution in complex with glucosaminyl(α1→6)-myo-inositol (GMI) and revealed the myo-inositol moiety of GMI occupying the same position as free myo-inositol, whereas the glucosamine moiety lying exposed to solvent at the entrance of the catalytic site (in “Heinz et al., (1995) EMBO J. 14: 3855-3863” and “Heinz et al., (1996) Biochemistry 35: 9496-9504). The residual portion of the core tetrasaccharide has little contact with the PI-PLC, which may explain the remarkable structural diversity of the core glycan within GPI anchors accepted. Taken together, current experimental data indicate that the catalytic mechanisms of cleavage of PI and GPI by bacterial PI-PLC as well as of GPI by trypanosomal GPI-PLC are all similar. However, it was not-known whether this is true for the adipocyte plasma membrane GPI-PLC, considering the failure of mammalian PI-PLC generating the second messenger, inositol-trisphosphate, to accept GPI anchors and of bacterial PI-PLC to cleave phosphorylated PI.
Substrate requirements of bacterial and trypanosomal (G)PI-PLC have been examined previously with PI analogs and GMI derivatives. Catalysis requires a free OH-group at the inositol-2 position. In studies using myristate-containing VSG from T. brucei, (G)PI-PLC was blocked competitively by 2-deoxy-inositol analogs of GMI, indicating that the inositol-2-OH is dispensable for substrate recognition, albeit required for catalysis. In addition, substrate recognition requires a charged phosphoryl group (i.e. phosphonate or phosphodiester) at the inositol-1 position (“Morris, J. C., et al., (1996) J. Biol. Chem. 271: 15468-15477”). Thus, the OH-groups at both the inositol-1 and inositol-2-positions are involved in catalysis, yet only the phosphoryl group appears to be needed for substrate recognition. Interestingly, glucosamine (α1→6)-inositol-1,2-cyclic phosphate turned out to be a better inhibitor than GMI-1-phosphate for trypanosomal GPI-PLC, but not the bacterial PI-PLC, indicating that the cyclic version may act as a product analog for the former. Furthermore, phosphonate derivatives of GMI-1-phosphate were found to be more potent inhibitors, most likely because they are non-cleavable substrate analogs. These data fit to the proposed two-step mechanism for bacterial PI-PLC action with PI first being cleaved to produce a cyclic inositol-1,2-phosphate (cIP) structure which is then hydrolyzed to inositol-1-phosphate. Interestingly, a cIP structure can be immunologically identified as so-called cross-reacting determinant in trypanosomal VSG upon exposure to GPI-PLC from T. brucei indicating operation of the first (cyclization) but not of the second step (decyclization) during trypanosomal GPI-PLC catalysis. The requirement for transient or stable cIP formation is consistent with the finding that GPI anchors with their inositol residue palmitoylated at the 1- or 2-position, such as that of human erythrocyte AChE, resist phospholipase cleavage.
GMI-1-dodecylphosphonate turned out to be considerably more inhibitory than the corresponding hexyl derivative for the trypanosomal GPI-PLC which is also membrane-associated (“Morris, J. C. et al. (1995) J. Biol. Chem. 270: 2517-2524”). Interestingly, it has been found that, in the absence of carbohydrate substituents (i.e. glucosamine) on the inositol, non-cleavable analogs of inositol-1-phosphate, as exemplified by myo-inositol-1-O-dodecylphosphate, inhibit the trypanosomal GPI-PLC. The efficacy of this type of inhibitor was considerably increased upon substitution of the 2-position of 2-deoxy-inositol-1-O-dodecylphosphonates with 2-fluoro substitutions competitively inhibiting trypanosomal GPI-PLC with IC50 of 10-90 μM (“Morris, J. C. et al., (1996) J. Biol. Chem. 271: 15468-15477”). The most potent inhibitors of GPI-PLC reported so far have both a fluoro group at the 2-position and a dodecyl-phosphonate at the 1-position of 2-deoxy-inositol being at least 5-fold more inhibitory than myo-inositol-1-O-dodecyl-phosphonic acid (Morris, J. C. et al. (1998) Biochem. Biophys. Res. Commun. 244: 873-867″). Interestingly, differential inhibition of (G)PI-PLC from B. cereus and T. brucei by some of these compounds argues that the two enzymes represent mechanistic subclasses of (G)PI-PLC.
Unfortunately, analogous data are not yet available for mammalian GPI-PLC. Surprisingly, the newly synthesized myo-inositol-1,2-cyclo-dodecylphosphonic acid (GPI-2350) turned out to be a potent inhibitor of bacterial as well as adipocyte GPI-PLC.
The initial observation that alkaline phosphatase (aP) was released from the membrane bilayer by a bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) led to the identification of another type of membrane attachment for proteins involving the covalent coupling to a glycosylphosphatidylinositol (GPI) lipid.
The first complete structure of a GPI anchor was elucidated for the variant surface glycoprotein (VSG) from Trypanosome brucei. 
The core tetrasaccharide consists of three mannose residues and a non-acetylated glucosamine, one end of which is amide-linked to the protein moiety via a phosphoethanolamine bridge and the other end of which is glycosidically linked to the 6-hydroxyl group of phosphatidylinositol (PI). PI is cleaved by (G)PI-specific phospholipases of specificity C and D ([G]PI-PLC/D) releasing diacylglycerol and phosphatidic acid, respectively, and leaving a terminal (phospho)inositolglycan (PIG) structure at the protein moiety.
Since then, bacterial PI-PLC of various origin have been commonly used to detect GPI-anchored proteins (GPI-proteins).
Lipolytic release of the protective surface coat consisting of GPI-anchored VSG by GPI-PLC is assumed to be required for T. brucei to achieve antigenic variation in order to escape the immune system of the host.
Since most mammalian cells and tissues express GPI-proteins, the majority of them with their GPI anchor embedded in the outer leaflet of the plasma membrane, endogenous (G)PI/PI-PLC/D may control the specific down-regulation of their cell surface expression and simultaneously the increase of the soluble protein moiety in the circulation.
Soluble forms of GPI-proteins have been detected circulating within the blood stream, such as 5-nucleotidase (5′-Nuc), Thy-1, alkaline Phosphatase (aP), and CD16 receptors. GPI-PLC could be also identified in human neutrophils, bovine brain, rat intestine and a human carcinoma cell line. GPI-PLC from rat liver has been purified to homogeneity. An endogenous GPI-PLC has been described as having the capability of releasing renal dipeptidase from porcine maximal tubules. However, elucidation of a GPI-PLC structure or gene is still missing. Whereas the mammalian GPI-PLC is membrane associated, the mammalian GPI-PLD can be recovered from all types of tissue material of different species (human, rat, bovine) as well as organs (placenta, brain, liver, serum).
The main role of GPI-PLD is probably the degradation of the GPI anchor of GPI proteins after endocytosis and trafficking to lysosomes. Mammalian tissues harbor two distinct GPI-PLD, being active in serum and at the cell surface with different functionality.
Some of the GPI-proteins, such as 5′-Nuc and aP in yeast and rodent adipocytes, do not seem to be released as soluble versions from the cell surface upon lipolytic cleavage of their GPI anchors by a GPI-PLC, both in vitro and in vivo.
Additional mild salt and/or trypsin cleavage is required for recovery of the protein moieties of some lipolytically cleaved GPI-proteins in the soluble fraction/medium after separation from the particulate fraction/cells—indicative of the existence of a receptor protein.
In these cases, the activity of GPI-PLC does not affect the localization or topology of GPI-proteins, but does, instead, modify their functional (catalytic/binding) characteristics in the course of their conversion from the amphiphilic into the hydrophilic version. The presence of an intact GPI anchor affects the conformation and behavior of the protein moiety attached to it.
In case of 5′-Nuc and Gce1, the catalytic and binding efficiency was increased in the lipolytically cleaved GPI-protein when compared to the intact form embedded in the membrane or reconstituted into detergent micelles or liposomes.
GPI-PL is up-regulated in eucaryotes by nutritional signals, as, e.g., glucose in yeast, where lipolytic processing of GPI-proteins seems to play a role during biogenesis of the cell wall, and by glucose as well as certain hormones, growth factors and drugs (e.g. insulin, glimepiride) in rodent adipocytes, myocytes and human endothelial cells.
Glimepiride, an antidiabetic drug, lowers blood glucose predominantly by stimulating the insulin release from pancreatic cells and, additionally, but to a minor degree, by mimicking metabolic insulin action in peripheral tissues, such as activation of glucose transport in muscle cells and inhibition of lipolysis in adipocytes.
The blood glucose-lowering effect of the sulfonylurea, glimepiride, is partly caused by stimulation of non-oxidative glucose metabolism in adipose and muscle cells via insulin receptor-independent activation of the IRS-PI3K pathway. In isolated rat adipocytes, the molecular mechanism of glimepiride action has been demonstrated to involve the redistribution and concomitant activation of lipid raft-associated signaling components, such as the acylated non-receptor tyrosine kinase, pp59Lyn, and some GPI-proteins, as well as the stimulation of a plasma membrane glycosylphosphatidylinositol-specific GPI-PLC, which is also moderately activated by insulin.
Glimepiride is a sulphonylurea agent that stimulates insulin release from pancreatic-β-cells and may act via extrapancreatic mechanisms. It is administered once daily to patients with type 2 (non-insulin-dependent) diabetes mellitus in whom glycaemia is not controlled by diet and exercise alone, and may be combined with insulin in patients with secondary sulphonylurea failure.
The greatest blood glucose lowering effects of glimepiride occur in the first 4 hours after the dose. Glimepiride has fewer and less severe effects on cardiovascular variables than glibenclamide (glyburide). Pharmacokinetics are mainly unaltered in elderly patients or those with renal or liver disease. Few drug interactions with glimepiride have been documented.
In patients with type-2 diabetes, glimepiride has an effective dosage range of 0.5 to 8 mg/day, although there is little difference in efficacy between dosages of 4 and 8 mg/day. Glimepiride was similar in efficacy to glibenclamide and glipizide in 1-year studies. However, glimepiride appears to reduce blood glucose more rapidly than glipizide over the first few weeks of treatment. Glimepiride and gliclazide were compared in patients with good glycaemic control at baseline in a 14-week study that noted no differences between their effects. Glimepiride plus insulin was as effective as insulin plus placebo in helping patients with secondary sulphonylurea failure to reach a fasting blood glucose target level of ≦7.8 mmol/L, although lower insulin dosages and more rapid effects on glycaemia were seen with glimepiride.
Although glimepiride monotherapy was generally well tolerated, hypoglycaemia occurred in 10 to 20% of patients treated for ≦1 year and ≦50% of patients receiving concomitant insulin for 6 months. Pooled clinical trial data suggest that glimepiride may have a lower incidence of hypoglycaemia than glibenclamide, particularly in the first month of treatment. Dosage is usually started at 1 mg/day, titrated to glycaemic control at 1- to 2-week intervals to a usual dosage range of 1 to 4 mg/day (maximum 6 mg/day in the UK or 8 mg/day in the US).
Glimepiride lowers glucose predominantly by stimulation of insulin release from pancreatic β-cells and, to a minor degree, by mimicking metabolic insulin action in peripheral tissues, such as activation of glucose transport in muscle cells and inhibition of lipolysis in adipocytes.
Glimepiride has been demonstrated to potently induce the amphiphilic-to-hydrophilic conversion of a subset of GPI-proteins, such as 5′-Nuc, aP, and Gce1, by activation of a GPI-PLC upon treatment of primary or cultured rodent adipocytes with pharmacological concentrations.
The inositol derivative, GPI-2350, which is disclosed the first time in this invention, inhibits bacterial, trypanosome and serum GPI-PLC and GPI-PLD with high potency (IC50=0.2-10 μM) and selectivity. GPI-2350 almost completely down regulates the GPI-PLC in intact rat adipocytes. Whereas GPI-PLC plays no role in metabolic insulin signaling, the activation of GPI-PLC is indispensable for the insulin-mimetic effects of glimepiride via the insulin receptor-independent cross-talk from detergent insoluble glycolipid-enriched lipid raft domains (DIGs) to the insulin receptor substrate-1 (IRS-1).
DIGs, which are expressed in high number in the plasma membrane of many terminally differentiated cells, such as adipocytes, are special membrane microdomains which serve as platform for membrane-mediated biological processes, including signal transduction and trafficking and sorting of proteins and lipids. They are enriched in cholesterol and (glyco)sphingolipids in the exoplasmic leaflet and in phospholipids with saturated acyl chains and cholesterol in the inner leaflet, forming a liquid-ordered phase within the bilayer. DIGs are characterized by insolubility in 1% Triton X-100 in the cold and low buoyant density upon sucrose gradient centrifugation. Based on these criteria, certain GPI-anchored, acylated and transmembrane signaling proteins have been found to be enriched in DIGs vs. non-DIG areas of the plasma membrane. Furthermore, DIGs of higher (hcDIGs) and lower cholesterol (IcDIGs) content can be distinguished from one another on the basis of their lower and higher buoyant density, respectively.
The stimulus-dependent redistribution of certain GPI-anchored as well as acylated signaling proteins from hcDIGs to IcDIGs was blocked by GPI-2350.
GPI-2350 reduced the basal and glimepiride/insulin-induced lipolytic release of GPI-proteins, such as Gce1 and 5′-Nuc, from intact rat adipocytes by lipid raft-associated GPI-PLC (IC50=5-10 μM). Inhibition of the GPI-PLC by GPI-2350 (50 μM) led to almost complete blockade of (i) the dissociation from caveolin of pp59Lyn and Gce1, (ii) their redistribution from hcDIGs to IcDIGs, (iii) tyrosine phosphorylation of pp59Lyn and IRS-1, (iv) stimulation of glucose transport and (v) inhibition of lipolysis in response to glimepiride.
Insulin activation of the GPI-PLC had a moderate effect on lipid raft distribution; and its minor role, if any, in metabolic insulin signaling was demonstrated in the presence of GPI-2350 only, since it (e.g. tyrosine phosphorylation of IRS-1 and inhibition of lipolysis) was only marginally reduced.
Lipolytically cleaved GPI-proteins generated by the glimepiride-induced GPI-PLC remain associated with hcDIGs rather than redistribute to IcDIGs, as do their uncleaved amphiphilic versions as well as pp59Lyn.
The cross-talk of glimepiride to the insulin signaling cascade via IRS tyrosine phosphorylation by redistributed and activated pp59Lyn in rat adipocytes requires activation of the hcDIGs-associated GPI-PLC.